The sensory receptors of the inner ear, the hair cells, mediate our senses of hearing and balance because of their ability to convert mechanical forces into electrical signals. This process happens in the mechanosensitive organelle, the hair bundle, which protrudes from the cell's apical surface. The hair bundle is composed of a few tens to hundreds of actin-rich stereocilia that are arranged in a hexagonal array. Mechanical deflection of the bundle toward the tallest stereocilia leads to shearing motions between adjacent stereocilia that are exerted by tip links, connectors between the tips of shorter stereocilia with its taller neighbor. The consequential increase of mechanical tension in the transduction apparatus increases the open probability of mechanically gated ion channels located at or near the tips of stereocilia, resulting in an influx of cations that depolarizes the hair cell, thereby generating a receptor potential. The understanding of hair bundle development, maintenance, and ultimately the molecular basis of its function, is fundamental for inner ear biology and also for our understanding of human hearing loss and disequilibrium. One aim of this proposal is to investigate the molecular basis of stereocilia height regulation by investigating the consequences of loss of the gene encoding twinfilin 2, a protein that is located near the tips of stereocilia. For this purpose, we will employ transgenic mouse technology, quantitative 3-dimensional microscopic analyses using confocal and electron microscopy, as well as electrophysiology. A second aim focuses on the role of a group of TRP proteins including members of the TRPML and TRPV subfamily that appear to be able to form heteromeric ion channels in hair cells. We hypothesize testing whether these heteromeric channels are functional, and whether they play a role in hair cell function. Particularly, we are interested in exploring a role of TRPML/TRPV heteromers in mechanoelectrical transduction. Analyses include biochemical, electrophysiological, and cell-based assays, as well as transgenic mice, histology, whole animal auditory function tests, and electrophysiology. PUBLIC HEALTH RELEVANCE: In this grant application, it is proposed to investigate the role of twinfilin 2, a newly identified hair bundle protein, in hair bundle development and in mature hair cells. In an independent aim, it is proposed to test whether TRPML3 and the related TRP channels TRPML2, TRPV5, and TRPV6 play important roles in hair cells or other inner ear cell types. Both topics are being pursued using transgenic mouse technology, high-resolution imaging, and electrophysiology. Understanding hair bundle development and the functional role of specific ion channels in inner ear cell types is fundamental for inner ear biology and also for our understanding of human hearing loss and disequilibrium.